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In a recent study posted to the medRxiv* preprint server, researchers pursued two monkeypox virus (MPXV) immune targets, A35R and H3L, that could be deployed in a serological assay to identify recent MPXV cases.

Study: A35R and H3L are Serological and B Cell Markers for Monkeypox Infection. Image Credit: Berkay Ataseven/Shutterstock


The 2022 MPXV outbreak is the largest recorded outbreak in non-endemic countries. As of August 15, 2022, it has infected over 35000 people outside Africa, hydrea iv with most cases diagnosed in Spain and the United States (US). The MPXV case mortality rate has remained under 0.5% during the current outbreak. 

Currently,  the MPXV diagnosis relies on polymerase chain reaction (PCR) that detects the MPXV nucleic acids. However, as infection rates continue to surge, there is an urgent need for more rapid and sensitive diagnostic tests, such as rapid antigen-based assays, that could be performed on point-of-care (POC) sites. Moreover, serological assays offer other additional benefits. They can promote the understanding of both T cell and B cell responses and lead to the isolation of neutralizing antibodies (nAbs) for later examination as therapeutics. Lastly, serological assays can highlight potential targets for vaccine candidates.

Previous experiments on the MPXV-related Vaccinia virus (VACV) identified proteins needed for viral attachment and entry, many of which were targeted by nAbs, elicited in both infected and vaccinated humans. However, studies have neither identified nor characterized the main serological and B cell markers accompanying MPXV infection. Like other pox viruses, MPXV adopts two forms, mature virion (MV) and enveloped virion (EV), and displays distinct proteins on its surface during intra- and inter-host transmission, depending on those two forms. While A35R is one of the six proteins expressed on the EV form and mediates cell-to-cell viral spread, H3L antigen, expressed on the MV form, promotes binding to host cells and infectivity.

About the study

In the present study, researchers tested the reactivity of four MPXV antigens, two from the MV and EV each, against sera obtained from 11 MPXV convalescent donors in Israel. These donors donated sera 33 to 62 days post-infection (pi) between May and June 2022 to help the researchers investigate the nAbs elicited following natural MPXV infection. The two control groups comprised five male donors below 40 years and five over 50 years, all uninfected with MPXV, with the latter having received a VACV vaccine.

The team generated soluble domains of four MPXV antigens to map the targets for nAbs elicited following MPXV infection. The two EV form-derived antigens were A35R and A36R, and the two MV form-derived antigens were M1R and H3L, all cloned into the plasmid cloning deoxyribonucleic acid (pcDNA)3.1(-) expression vector. Further, they expressed these antigens in mammalian Expi293F cells and purified them on Nickel beads. The purified antigen was used to coat enzyme-linked immunosorbent assay (ELISA) plates and react with sera immunoglobulin G (IgG) from the MPXV convalescent donors and control groups.

First, the team performed an ELISA to test the binding of IgG in heat-inactivated MPXV convalescent sera to heat-inactivated VACV-coated plates. They also performed a plaque reduction neutralization assay (PRNA) to determine whether IgG from MPXV convalescent sera also neutralized the Vaccinia Lister virus (VACV Lister). The researchers incubated 50 plaque-forming units (PFU) per well of VACV Lister for one hour with purified IgG at a starting concentration of 200 μg/ml, followed by six consecutive two-fold dilutions to infect 5×105 Vero cells. They determined a 50% neutralization titer (NT50) 72 hours pi.

Lastly, the researchers evaluated whether donor B cells specifically bound A35R and H3L antigens. This experiment collected five to six million peripheral mononuclear blood cells (PBMCs) and stained them for the cluster of differentiation (CD)3, CD19, and IgG. Additionally, they tested them against biotinylated, streptavidin fluorophore-conjugated A35R and H3L.

Study findings

The authors noted that IgG from heat-inactivated sera of all MPXV convalescent donors bound VACV. Conversely, sera from under 40 years of uninfected males (control group 1) had minimal reactivity to the heat-inactivated VACV. However, that from the control group 2 (>50 years) recognized VACV, although with much-reduced sensitivity than MPXV convalescent samples, demonstrating that vaccinated individuals exhibit high nAb titers even after 50 years of vaccination.

Notably, only four MPXV convalescent donors exhibited neutralizing activity. Although the authors noted a negative correlation between NT50 values and the time from infection to sample collection, it was statistically insignificant likely due to the small sample size. This finding clarified that though MPXV infection induced strong serological responses in ELISA, most recovered patients did not have nAbs against VACV Lister within 33 to 62 days after MPXV infection. Interestingly, sera IgG from all MPXV convalescent donors bound A35R in a dose-dependent manner, whereas both control groups exhibited significantly lower binding to A35R. The researchers also detected MPXV convalescent sera binding to H3L antigen, albeit with a weaker signal than A35R. Notably, MPXV convalescent donors and control group 2 showed similar H3L responses.


Overall, the study results revealed that although nAbs elicited following MPXV infection targeted both A35R and H3L antigens, the 124-amino acid truncation of A35R distinguished between recent MPXV infection and past VACV vaccination. Likewise, a 276-amino acid truncation of the H3L antigen also distinguished between recent MPXV illness and prior VACV vaccination. Additionally, all 11 MPXV donors displayed A35R-specific B cells, and eight of 11 donors also exhibited H3L-specific B cells. Therefore, recombinant soluble truncated forms of A35R and H3L proteins could be used in ELISA to diagnose recent cases of MPXV infections.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Ron Yefet, Nadav Friedel, Hadas Tamir, Ksenia Polonsky, Michael Mor, David Hagin, Eli Sprecher, Tomer Israely, Natalia T Freund. (2022). A35R and H3L are Serological and B Cell Markers for Monkeypox Infection. medRxiv. doi: https://doi.org/10.1101/2022.08.22.22278946 https://www.medrxiv.org/content/10.1101/2022.08.22.22278946v2

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: Amino Acid, Antibodies, Antigen, Assay, B Cell, Blood, Cell, Cloning, Diagnostic, ELISA, Enzyme, Fluorophore, heat, Immunoglobulin, Monkeypox, Mortality, Plasmid, Polymerase, Polymerase Chain Reaction, Therapeutics, Vaccine, Vaccinia Virus, Virus

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Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.

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